Development of robust, sensitive, and reusable sensors is a strong current scientific priority. As such, recognition-based biosensors capable of specifically detecting chemicals, toxins, and bio-agents in their environment are of increasing importance. An important goal in biosensor evolution is production of nanoscale assemblies capable of continuously monitoring concentrations of target species in a simple, reliable manner. This is accomplished by designing sensor components to carry out analyte recognition and binding while simultaneously producing useful output signals via an integrated signal transduction system. Optically addressed biosensors of this type often employ fluorescence resonance energy transfer (FRET) in signal transduction.
FRET has been employed in carefully designed sensing systems for proteins, peptides, nucleic acids and other small molecules, see Medintz et al., Nature Materials 2003 2, 630. It is known in the art that other sensing modalities can be employed in the signal read-out of recognition-based biosensors, especially electrochemical modes or enzyme related systems, see Benson et al., Science 293, 1641-1644 (2001).
Biosensors function by reversibly linking bioreceptor-target analyte binding with closely integrated signal generation. Such sensors can either continuously monitor analyte concentrations or easily be returned to baseline read-out values by removal of analyte. Current bioassays on the market are single use or limited time use. Either they need to be replaced after each test or within a short time. This increases both test costs and the logistical demands for performing the analysis. Fielded biosensors can have complex robotics that handle the reagent storage and sensor surface replacement.
Sensor systems based on Surface Plasmon Resonance (SPR) can be regenerated, such as the Biacore SPR instrument. This sensor works by measuring the change in index of refraction at the sensor surface upon analyte binding. This works well for large molecules, but requires a harsh regeneration fluid that limits sensor lifetime. In addition, it works poorly in complex samples where nonspecific deposition to the surface interferes with the ability to discriminate actual signal.
Other systems can be used numerous times for the detection of small molecules, however as the fluorescent analog is displaced off the sensor surface the sensor is slowly consumed until it no longer functions. One example is the flow immunosensor of U.S. Pat. No. 5,183,740 to Ligler, et al.
Surface acoustic wave sensors with selective membranes exist for the sensitive detection of gas phase molecules. However, similar devices built for the liquid phase detection are less sensitive and have the same limitations as SPR.
FRET based assays have previously been described; however they are fluid phase methods that are effective for a single analysis only. This is also true for tests that depend on fluorescence anisotropy measurements, which are effective solution phase analyses but require the reagents to be freely moving to monitor a change upon binding.
A definition describing a biosensor has been proposed by IUPAC which provides that “a biosensor is a self-contained integrated device which is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element (biochemical receptor) which is in direct spatial contact with a transducer element. A biosensor should be clearly distinguished from a bioanalytical system which requires additional processing steps, such as reagent addition. Furthermore, a biosensor should be distinguished from a bioprobe which is either disposable after one measurement, i.e. single use, or unable to continuously monitor the analyte concentration”. Although biological recognition elements are employed in an extensive range of analytical formats, in few cases are they integrated into sensing devices and meet all these rigorous criteria.
The functional simplicity afforded by biosensors, allowing autonomous and continuous monitoring of chemical species, promises to make these devices useful in chemical process monitoring, pharmaceuticals screening, patient point-of-care and environmental testing, public health, and in defense-related fields.
Optically addressed molecular biosensors that meet the above criteria have been developed by Hellinga et al., Proc. Natl. Acad. Sci. USA 1997, 94, 4366-4371, in which bacterial periplasmic binding proteins (bPBPs) were engineered to allow transduction of binding events to remote fluorescent signal-generating sites within the same protein by allosteric coupling. Recently, this strategy has been extended to allow surface tethering to unmodified hydrophobic surfaces of dye labeled-bPBPs by engineering of a self-adhering hydrophobic peptide onto the protein terminus, see Wada, et al, J.A.C.S. 2003, 52, 16228-16234.
FRET-based fusion protein biosensors that employ different colored green fluorescent protein (GFP) mutants linked to substrate binding domains that report binding events by coupled changes in conformation and energy transfer have also been developed, see Fehr, et al, Proc. Natl. Acad. Sci. USA 2002, 99, 98469851, and Fehr et al, J. Biological Chem. 2003, 278, 19127-19133. Both of these biosensor types are only  useful for a small range of analysis targets. In the bPBP-based sensors, intramolecular transduction of binding events to integrated signaling centers requires highly specialized allosteric receptors. Even though computationally intensive redesign of the binding sites for recognition of alternate substrates may be feasible, binding pocket remodeling is unlikely to prove practical in providing sensors useful for monitoring a wide range of analytes. In sensors employing GFP fusion proteins, where differences in FRET efficiency between bound- and analyte-free receptor states result in signal generation, the range of bioreceptors that undergo obligatory ligand-dependent conformational changes is also very limited.
A variety of sensitive FRET-based single-measurement bioanalytical systems or bioprobes have been developed that detect peptides, proteins, and various small molecules in vivo and in vitro. See Scheller, et al, Biotech. 2001, 12, 35-40; Iqbal, et al, Biosens. Bioelect. 2000, 15, 549-578; O'Connell, et al, Anal. Lett. 2001, 34, 1063-1078; and Marvin, et al Proc. Natl. Acad. Sci. USA 1997, 94, 4366-4371. Maxwell, et al, J. Am. Chem. Soc. 2002, 124, 9606-9612, describes a gold nanoparticle-nucleic acid FRET biosensor that utilizes a probe DNA oligo in a ‘molecular beacon’ function to detect other DNA.
Biosensors that utilize FRET are also attractive due to the intrinsic sensitivity of FRET to small changes in donor-acceptor distance and orientation. Medintz, et al, Bioconjugate Chem. 2003, 14, 909-918 demonstrated the feasibility of using dye-labeled MBP and dye-labeled β-CD for FRET-based detection in solution. Rather than being a sensor, that homogenous system functions only in single-measurement bioanalysis.